Seminal fibronectin type-II (Fn-II) proteins interact with choline phospholipids present on the sperm plasma membrane and play a crucial role in sperm capacitation. Crystal structure of phosphorylcholine (PrC) complex of PDC-109, the major bovine Fn-II protein, together with fluorescence spectroscopic studies has shown that tryptophan residues are crucial for its specific interaction with choline phospholipids. In the present study, the heterogeneity and microenvironment of tryptophan residues in HSP-1/2, a major protein of horse seminal plasma (which is homologous to PDC-109) were investigated in the native state, in the presence of PrC and phosphatidylcholines (PCs) with short (valeryl, C-5) and long (myristoyl, C-14) chains, and upon denaturation using fluorescence quenching, time-resolved fluorescence and red-edge excitation shift (REES) measurements. The results obtained show that the environment of tryptophan residues in HSP-1/2 is more heterogeneous as compared to that in PDC-109. Binding of choline containing ligands afforded a protection to the tryptophan residues with the shielding order being: PrC≤divalaroyl PC<dimyristoyl PC. REES value obtained for HSP-1/2 (3.5nm) is smaller than that of observed for PDC-109 (4nm) and binding to PrC and DVPC reduced the REES to 1nm. HSP-1/2 exhibits only partial unfolding with chemical denaturants with no cooperativity, whereas complete unfolding was observed in the presence of 10mM dithiothreitol, indicating that disulfide linkages prevent complete unfolding of the protein. In the presence of PrC the transition midpoints shifted to higher concentrations of the denaturant together with a broadening of the sigmoidal transitions, indicating that ligand binding as well as polydispersity modulate the unfolding process.
Keywords: Capacitation; Chaperone; Chemical unfolding; Fluorescence quenching; Polydispersity; Red edge excitation shift.
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